Pulsed EPR dipolar spectroscopy at Q- and G-band on a trityl biradical

Post-print (lokagerð höfundar)Pulsed electron paramagnetic resonance (EPR) spectroscopy is a valuable technique for the precise determination of distances between paramagnetic spin labels that are covalently attached to macromolecules. Nitroxides have commonly been utilised as paramagnetic tags for biomolecules, but trityl radicals have recently been developed as alternative spin labels. Trityls exhibit longer electron spin relaxation times and higher stability than nitroxides under in vivo conditions. So far, trityl radicals have only been used in pulsed EPR dipolar spectroscopy (PDS) at X-band (9.5 GHz), K-u-band (17.2 GHz) and Q-band (34 GHz) frequencies. In this study we investigated a trityl biradical by PDS at Q-band (34 GHz) and G-band (180 GHz) frequencies. Due to the small spectral width of the trityl (30 MHz) at Q-band frequencies, single frequency PDS techniques, like double-quantum coherence (DQC) and single frequency technique for refocusing dipolar couplings (SIFTER), work very efficiently. Hence, Q-band DQC and SIFTER experiments were performed and the results were compared; yielding a signal to noise ratio for SIFTER four times higher than that for DQC. At G-band frequencies the resolved axially symmetric g-tensor anisotropy of the trityl exhibited a spectral width of 130 MHz. Thus, pulsed electron electron double resonance (PELDOR/DEER) obtained at different pump-probe positions across the spectrum was used to reveal distances. Such a multi-frequency approach should also be applicable to determine structural information on biological macromolecules tagged with trityl spin labels.The authors acknowledge Dr Vasyl Denysenkov for the technical support with the G-band EPR spectrometer and Dr Alice Bowen for useful discussions and for proof reading the manuscript. This work is supported by SPP 1601 New Frontiers in Sensitivity for EPR Spectroscopy: from Biological Cells to Nano Materials from the German Research Society DFG, the Cluster of Excellence Frankfurt (CEF) Macromolecular Complexes and the Icelandic Research Fund (120001021), which are all gratefully acknowledged.Peer reviewe

Sterically shielded spin labels for in-cell EPR spectroscopy: Analysis of stability in reducing environment

Post-print (lokagerð höfundar)Electron paramagnetic resonance (EPR) spectroscopy is a powerful and widely used technique for studying structure and dynamics of biomolecules under bio-orthogonal conditions. In-cell EPR is an emerging area in this field; however, it is hampered by the reducing environment present in cells, which reduces most nitroxide spin labels to their corresponding diamagnetic N-hydroxyl derivatives. To determine which radicals are best suited for in-cell EPR studies, we systematically studied the effects of substitution on radical stability using five different classes of radicals, specifically piperidine-, imidazolidine-, pyrrolidine-, and isoindoline-based nitroxides as well as the Finland trityl radical. Thermodynamic parameters of nitroxide reduction were determined by cyclic voltammetry; the rate of reduction in the presence of ascorbate, cellular extracts, and after injection into oocytes was measured by continuous-wave EPR spectroscopy. Our study revealed that tetraethyl-substituted nitroxides are good candidates for in-cell EPR studies, in particular pyrrolidine derivatives, which are slightly more stable than the trityl radical.This work was supported by the Icelandic Research Fund (120001021), the Deutsche Forschungsgemeinschaft (SFB 902, Molecular principles of RNA-based regulation) and by a doctoral fellowship to A. P. Jagtap from the University of Iceland Research Fund.Peer reviewe

A modular approach for the synthesis of nanometer-sized polynitroxide multi-spin systems

S.V. is supported by an EPSRC Doctoral Training Grant; B.E.B. acknowledges an EaStCHEM Hirst Academic Fellowship by the School of Chemistry, St Andrews and funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (REA 334496)The synthesis of rigid symmetric polyradical model systems with inter-spin distances between 1.4 and 4 nm and their room temperature continuous wave (CW) EPR spectra are reported. Conditions for attachment of the spin-label via esterification have been optimized on the direct synthesis of polyradicals from commercially available polyphenols and the carboxylic acid functionalized nitroxide TPC. A common synthetic protocol utilizing 4-hydroxy-4′-iodobiphenyl as a key building block has been used to synthesize an equilateral biradical and a triradical in only two steps from commercially available starting materials. The first synthesis of a tetraradical based upon an adamantane core bearing six equivalent nitroxide–nitroxide distances is also reported. These systems are very promising candidates for studying multi-spin effects in pulsed EPR distance measurements.Publisher PDFPeer reviewe

Mimicking the Intramolecular Hydrogen Bond: Synthesis, Biological Evaluation, and Molecular Modeling of Benzoxazines and Quinazolines as Potential Antimalarial Agents.

The intramolecular hydrogen bond formed between a protonated amine and a neighbouring H-bond acceptor group in the side chain of amodiaquine and isoquine is thought to play an important role in their antimalarial activities. Here we describe isoquine-based compounds in which the intramolecular H-bond is mimicked by a methylene linker. The antimalarial activities of the resulting benzoxazines, their isosteric tetrahydroquinazoline derivatives, and febrifugine-based 1,3-quinazolin-4-ones were examined in vitro (against Plasmodium falciparum) and in vivo (against P. berghei). Compounds 6b,c caused modest inhibition of chloroquine transport via the parasite’s ‘chloroquine resistance transporter’ (PfCRT) in a Xenopus laevis oocyte expression system. In silico predictions and experimental evaluation of selected drug-like properties were also performed on compounds 6b,c. Compound 6c emerged from this work as the most promising scaffold of the series; it possessed low toxicity and good antimalarial activity when administered orally to P. berghei-infected mice

Mimicking the Intramolecular Hydrogen Bond: Synthesis, Biological Evaluation, and Molecular Modeling of Benzoxazines and Quinazolines as Potential Antimalarial Agents

The intramolecular hydrogen bond formed between a protonated amine and a neighboring H-bond acceptor group in the side chain of amodiaquine and isoquine is thought to play an important role in their antimalarial activities. Here we describe isoquine-based compounds in which the intramolecular H-bond is mimicked by a methylene linker. The antimalarial activities of the resulting benzoxazines, their isosteric tetrahydroquinazoline derivatives, and febrifugine-based 1,3-quinazolin-4-ones were examined in vitro (against Plasmodium falciparum ) and in vivo (against Plasmodium berghei ). Compounds 6b,c caused modest inhibition of chloroquine transport via the parasite's "chloroquine resistance transporter" (PfCRT) in a Xenopus laevis oocyte expression system. In silico predictions and experimental evaluation of selected drug-like properties were also performed on compounds 6b,c. Compound 6c emerged from this work as the most promising analogue of the series; it possessed low toxicity and good antimalarial activity when administered orally to P. berghei -infected mice